Blood to be analyzed for diagnostic and monitoring purposes is customarily collected by venipuncture through a special cannula or needle attached to a syringe or evacuated collection tube. Such collection techniques and devices must offer ease and flexibility of use because of the large number of blood specimens that are processed and because of requirements for additives, variable volumes and adaptation to individual medical conditions.
Separation of the constituent phases, serum or plasma from the cells, is often necessary for laboratory analysis and is usually carried out by centrifugation or, occasionally, by filtration. This separation may require fractionation of minor as well as major components. Once separated the phases are best kept in an inert container, physically and chemically isolated, to avoid disturbance of analyte concentrations. It may be necessary to store them under controlled environmental conditions of temperature, atmosphere or light. The blood and its fractions have characteristics of volume, color and turbidity, of which it is important the analyst take note, since these may affect subsequent analyses. The blood may contain infectious agents and should be kept isolated, preferably in a closed system to reduce exposure to laboratory personnel. Blood specimens may be processed either in small numbers in physicians' offices where compactness and simplicity of use are required or in large numbers in clinics and hospitals where efficiency and assured identification are essential and automation is desirable.
Thus there is a need for a blood collection and separation device or system which combines the features of: ability to separate the blood phases under conditions which limit personnel exposure; maintenance of these phases separated and unchanged; monitoring of gross characteristics of the phases; ready adaptability to varying blood collection requirements; and flexibility for stand-alone use or integration into automated systems.
Serum and plasma are commonly used analytical samples. If serum is desired the specimen must be permitted to clot or coagulate before further separation is attempted. Activation of this clot formation may result as a consequence of contact with the glass collection tube in which the blood was collected and can be enhanced by the addition of various clot-activating materials as described in U.S. Pat. No. 4,189,382 by Zine. If plasma is desired the specimen must have an anticoagulant mixed with it immediately after collection. For this purpose such anticoagulant materials are commonly placed in blood collection devices at the time of manufacture.
The most commonly used blood collection devices are evacuated tubes. They are characterized by advantages and disadvantages in certain situations. The pre-evacuated blood collection tube (such as described by Kleiner U.S. Pat. No. 2,460,641) has the following advantages: once sterilized, its interior remains sterile without additional packaging; simplicity of structure and use, in that its basic form consists of only a glass tube permanently closed at one end with a rubber stopper in the open end; and it is self-sealing when blood drawing is complete and the cannula which was used to puncture the rubber stopper has been removed.
The blood plasma or serum phase is readily separated from the blood cells or clot phase by centrifugation since the specific gravities of these two phases are different. The recommended and usual practice is to centrifuge the specimen at a relative centripetal acceleration of 1000 to 1200 gravities for about 10 minutes. Various materials and devices have been described to physically separate the serum or plasma from the cellular phase, which are either activated during centrifugation or applied after separation is complete. These include gel-like compositions with densities intermediate to the phases as described, for example, in U.S. Pat No. 4,350,593 by Kessler and U.S. Pat. Nos. 3,852,194 and 4,083,784 by Zine. Such substances, as commonly used, are sealed in the evacuated blood collection tube at the time of manufacture and will migrate to, and form a barrier at, the interface between the blood phases under the influence of the correct centrifugal force. A problem with such materials is that although they are made from substances with low chemical reactivity they nevertheless contain substances which will contaminate the serum or plasma (such as low levels of some metals used as catalysts for the formation of those compositions). Some substances which are determined by blood analysis (such as low concentrations of organic-soluble, hydrophobic drugs) can be significantly adsorbed or absorbed out of the sample by such gel-like materials, resulting in incorrect analyses. Other separators consisting of a variety of plug-like objects have been used as described, for example, in U.S. Pat. No. 4,492,634 by Villa-Real, U.S. Pat. No. 3,508,653 by Coleman, U.S. Pat. No. 4,417,981 by Nugent, U.S. Pat. No. 4,425,235 by Cornell, and U.S. Pat. No. 4,369,117 by White. Unfortunately, these devices are more expensive to make and insert into the pre-evacuated collection tube and the barriers they provide are no more reliable or effective than the simpler, less expensive gel-like separation materials.
Irrespective of the relative expense of plug-like barrier devices, the main problem with such barriers of the prior art is that during blood collection it is impossible to keep blood from getting between the tube closure and the barrier device. Coleman, U.S. Pat. No. 3,508,653 describes a plug-like barrier which is removably attached to the stopper, but does not demonstrate how blood is prevented from being interposed between the stopper and plug-like barrier. In fact, he states that the plug need not be attached to the stopper, but only restrained from moving prior to centrifugation. Since Coleman's barrier must allow passage of fluid around it when pressure is applied, it follows that the space between the barrier and stopper, along with the rest of the tube, is evacuated prior to blood collection. If the space between the stopper and barrier is evacuated, then blood may forcibly fill the space between the two parts. This is entirely unacceptable because there is no certain way of isolating the cellular blood component of the blood located between the barrier and the stopper from the separated serum or plasma thus negating the effect of the barrier. Both Nugent U.S. Pat. No. 4,417,981 and Adler U.S. Pat. No. 3,929,646 try to address this problem by providing a path for the whole blood to move around and past the plug-like barrier during sample collection or centrifugation. However, in practice once blood interposed between the stopper and barrier has clotted, these passages are insufficient to ensure that the cellular component of the interposed blood will migrate to the other side of the barrier during centrifugation. Both Nugent and Cornell U.S. Pat. No. 4,425,235 try to address this problem by including a migrating gel in their plug-like barriers, but this negates the benefits of solid barriers over gel barriers. White, Pat. No. 4,369,117, avoids the problem by inserting his plug-like barrier into the collection tube after blood collection has occurred. This is not desirable because an additional, hazardous step is required in handling an open tube.
An additional problem with many barriers is incomplete isolation of the serum or plasma from the cellular phase. In the case of gel-like barriers, severe jarring as might occur if the sample is shipped or mailed to a testing laboratory, may disrupt the seal provided by the barrier. If the isolation provided by the barrier is incomplete or disrupted, interaction of the separated phases will cause inaccurate analytical results. Moreover, prolonged contact of the blood phases with a gel-like barrier separator will increase the degree of analytical error caused by interaction between the blood and the barrier. Therefore, with most such devices it is necessary to separate the phases soon after the blood is collected and then transfer the separated plasma or serum to another container for prolonged storage or transport. Problems which then arise are that the transferred sample can become incorrectly identified and that the process of transfer exposes the user to potentially hazardous or infectious blood.
A portion of the serum or plasma may be completely isolated after centrifugation by a device which is inserted into the open end of the collection tube and permits the one-way flow of serum from the collection tube into a separate sampling container through a filter which prevents any of the fibrin from passing into the serum or plasma sample. Fibrin in blood serum can cause blood analysis machines to clog; therefore, many clinical chemistry laboratories filter all serum as a precaution. Such filtering devices are described, for example, in U.S. Pat. No. 4,464,254 by Dojki, U.S. Pat. No. 3,929,646 by Adler, U.S. Pat. No. 4,602,995 by Cassaday and are manufactured and distributed under the name of "serum/plasma filter" by W. Sarstedt, Inc. It is possible to isolate the phases of blood with such a device so as to prevent diffusion of ions or other interaction between the phases. However, their use requires additional manipulation of the collection tube, consequent exposure of the user to the blood specimen and risk of contamination of the sample. Related devices employ multiple flexible containers with provision for flow of blood fractions from the collecting blood bag into a separate reservoir (for example, U.S. Pat. No. 4,447,220 by Eberle and U.S. Pat. No. 4,322,298 by Persidsky) but these are bulky complex systems only for the separation of anticoagulated blood and are not suitable for collection and preparation of samples for routine clinical analysis.
For most analysis of centrifuged blood samples it is necessary to dispense a portion of the sample to other containers such as analyzer sample cups. Presently, this is done a number of ways. The most common method is to remove the stopper and use a dropper pipette to transfer some of the sample from the open tube to the alternate container. This procedure is hazardous in that removal of the stopper may generate aerosols containing infectious agents, and handling an open sample tube introduces a danger of spilling the sample. Another popular method of dispensing a sample to additional containers is to remove the stopper and simply decant into the additional containers. This method is even more hazardous than the first because skill is required to decant a small amount of serum or plasma without spillage.
Some devices have been made which attempt to address these hazards. One such device is the Tip-Top TM Dispenser Cap made by Helena Laboratories of Beaumont, Tex. The Tip Top dispenser is fastened to the open end of a centrifuged blood collection tube, inverted, and then squeezed causing a portion of the sample to be dispensed through an orifice to a sample cup. The primary difficulty with the Tip Top dispenser and others like it is that it still requires the hazardous step of removing the stopper of the blood collection tube. A device which does not require stopper removal for dispensing a blood sample is the CleanTech TM system made by Clean Tech SCI AG of Langenthal Switzerland. The CleanTech system consists of several components including a cannula to puncture the stopper, a machine to insert the cannula into the stopper, a pipette to access the sample through the stopper and a pump which fastens to the pipette to draw the sample from the tube. This device goes far to address the hazards of dispensing a sample, but it is a relatively complex device and requires several steps to use. In some situations the use of conventional centrifuges to separate serum or plasma from the cellular component of blood specimens is undesirable because it requires a large and expensive centrifuge, best suited for separating batches of several specimens simultaneously. This operation is inefficient when the serial analyses of single samples is urgently required. Time must also be taken to properly balance the centrifuge rotor to prevent excessive vibration which may damage the machine and specimens. An apparatus such as the "StatSpin" axial centrifuge, developed and manufactured by Norfolk Scientific, Inc. (Norwood, MA), can effect this separation on a single specimen more quickly, however, the technique employed by this apparatus is limited to anticoagulated blood, collected separately in a conventional blood collection device and transferred to a specialized centrifuge chamber containing gel-like separation material. Moreover this transfer increases the hazard of contamination or loss of the sample, misidentification, and exposure of the operator to potentially infectious material in the blood. The use of an additional container increases the cost of analyzing a sample.
Similar objections and disadvantages apply to the "ACR-90" centrifuge chamber, rotor and "Airfuge" drive manufactured and sold by Spinco Division of Beckman Instruments, Inc. (Palo Alto, Calif.). This rotor is dual chambered and intended for isolation of the large lipid particles from lipemic sera. At high rotational speeds (typically greater than 90,000 rpm) the plastic chamber deforms, permitting the less dense lipid phase to migrate to a second chamber where it is trapped. Other axially spun centrifuge rotors, with a single volume often divided by vanes, are well known as "zonal rotors" and used for harvesting particles from a large volume (0.3-1.7 liters) of dilute solution such as preparations for vaccine by virologists and other such macromolecular isolates (Anderson, N. G.: Preparative zonal centrifugation. Methods of Biochemical Analysis 1967; 15: 271-310). Zonal rotors may be loaded and unloaded through a rotating seal while spinning (dynamically). A majority cannot be loaded or unloaded statically, while a few cannot be loaded or unloaded dynamically. In either case they are usually used for ultracentrifugation at rotational speeds of 20,000-60,000 rpm. Fluids are loaded by a pump and unloaded from them by displacement with air or a denser fluid pumped in during rotation. A single chamber, axially spun, centrifuge rotor with a variable volume which can be used for separation of plasma from blood was described by Brown in U.S. Pat. No. 4,530,691. This is intended for preparation of blood fractions for therapeutic use and relies upon the fractionation by centrifugation and isolation of those fractions by release of pressure exerted by a spring-loaded movable mandrel upon a flexible chamber. In this way the higher density cellular components can be taken off from the outer radius and the plasma through the center through fluid conduits while the rotor is in motion. Neither of these technologies (zonal ultracentrifugation nor centrifuge with a movable mandrel) is suitable for the fractionation of blood specimens as normally required for clinical analyses. The volumes are too large; they require the use of anticoagulants and cannot be used with clotted whole blood; and they are not readily adapted for automated procedures.
Procedures for blood separation and analysis expose laboratory personnel to infectious agents that may be passed through contact with blood; e.g. hepatitis or acquired immune deficiency syndrome. In addition, conventional batch processing of blood specimen separation is labor-intensive and has not generally been automated whereas other processes in clinical laboratories have. Automation of blood separation can effectively isolate laboratory personnel from the dangers of blood processing while theoretically increasing the speed of the overall analytical procedure.
U.S. Pat. No. 4,828,716 and application Ser. Nos. 07/192,847 and 07/346,063 now U.S. Pat. No. 5,030,341 by McEwen et al described a method of separating a sample of blood contained in a tubular chamber wherein the tubular chamber and its contents are rotated about the chamber's longitudinal axis and providing a means of processing a sample of blood having the features of: ability to separate the blood phases under conditions which limit personnel exposure; maintenance of these phases separated and unchanged; monitoring of gross characteristics of the phases; ready adaptability to varying blood collection requirements; and flexibility for stand-alone use or integration into automated systems. The present invention provides an improved blood collection and separation device according to the invention of McEwen et al as described in U.S. Pat. No. 4,828,716 and U.S. patent application Ser. No. 07/192,847 and Ser. No. 07/346,063. U.S. patent application No. 07/033,769 now U.S. Pat. No. 4,828,716 and U.S. patent application Ser. No. 07/192,847 and Ser. No. 07/346,063 are herein incorporated by reference.